DIFFERENTIAL STIMULATION OF INOSITOL TRISPHOSPHATE ACCUMULATION IN CULTURED HUMAN ENDOTHELIAL CELLS BY THROMBIN
Vascular endothelial cells possess specific receptors for thrombin, and thrombin can interact with these receptors to activate the endothelial cells. However, the signal transduction mechanisms which mediate the cellular responses are not yet characterised. The aim of this study therefore, was to determine whether thrombin influenced the inositol phosphate transduction pathway in cultured human endothelial cells. Endothelial cells were isolated from both large and small vessels; these were human umbilical vein and the microvasculature of human omentum respectively. The endothelial cells stained positively with antibodies against Factor VIII antigen and another endothelial cell specific antigen (BMA 120). Pure human thrombin (0.1 - 10 units/ml) induced a dose-dependent formation of inositol phosphate, inositol biphosphate and inositol trisphosphate (IP3) in endothelial cells from large vessels prelabelled with tritiated inositol. The formation of IP3 was significantly increased after 15 sec., maximal after 1 min. and had returned almost to baseline levels after 4 min. This time course is consistent with its role as a second messenger. When the enzymic activity of thrombin was removed with phenylalanyl-prolyl-arginine chloromethyl ketone or d i i sopr opyIfluorophosphate, thrombin lost its ability to stimulate the accumulation of IP3. Thrombin at all concentrations tested was unable to stimulate the formation of IP3 in small vessel endothelial cells. However, IP3 formation could be stimulated by bradykinin (0.1-10 μM) in cells from both small and large vessels. The results demonstrate that active thrombin can induce the formation of IP3 in large vessel endothelium. But that there are differences in the way small vessel endothelium responds to thrombin.